This application is a national phase application of PCT/EP99/06912 filed Sept. 17, 1999, which claims priority to EP 98 11 7745.4 filed Sept. 19, 1998 and EP 98 11 7799.1 filed Sept. 18, 1998, and which are incorporated in their entirety by reference.
1. Field of the Invention
The present invention relates to a novel method for the amplification of DNA, this method being particularly useful for the amplification of the DNA or the whole genome of a single cell, chromosomes or fragments thereof. The present invention further relates to the application of the method in DNA analysis for medical, forensic, diagnostic or scientific purposes, like comparative genomic hybridization (CGH), fluorescence in situ hybridization (FISH), polymerase chain reaction (PCR), single strand conformation polymorphism analysis (SSCP), DNA sequence analysis, xe2x80x9closs of heterozygosityxe2x80x9d analysis (LOH), fingerprint analysis, and/or restriction fragment length polymorphism analysis (RFLP).
2. Description of the Related Art
Several documents are cited throughout the text of this specification. Each of the documents cited herein (including any manufacturer""s specifications, instructions, etc.) are hereby incorporated by reference; however, there is no admission that any document cited is indeed prior art of the present invention.
PCR (polymerase chain reaction) is an extremely powerful in vitro method for the amplification of DNA, which was initially introduced in 1985 (Saiki (1985), Science 230, 1350-1354). By repeated thermal denaturation, primer annealing and polymerase extension, PCR can amplify a single target DNA molecule to easily detectable quantities.
Although PCR was initially applied to amplify a single locus in target DNA, it is increasingly being used to amplify multiple loci simultaneously. Frequently used primers for this general amplification of. DNA are those based on repetitive sequences within the genome, which allow amplification of segments between suitable positioned repeats. Interspersed repetitive sequence PCR (IRS-PCR) has been used to create human chromosome- and region-specific libraries (Nelson (1989), Proc. Nati. Acad. Sci. USA 86, 6686-6690). In humans, the most abundant family of repeats is the Alu family, estimated to comprise 900,000 elements in the haploid genome, thus giving an average spacing of 3-4 kb (Hwu (1986), Proc. Natl. Acad. Sci. USA 83, 3875-3879). However, a major disadvantage of IRS-PCR is that repetitive sequences like Alu or L1 are not uniformly distributed throughout the genome. Alu elements, for example, are preferentially found in the light bands of human chromosomes. Therefore, such a PCR method results in a bias toward these regions while other regions are less represented and thus not amplified or an amplification can only be obtained below detectable levels. Furthermore, this technique is only applicable to those species where abundant repeat families have been identified, whereas other species such as Drosophila and less well characterized animals and plants cannot be subjected to this method.
A more general amplification than with ISR-PCR can be achieved with xe2x80x9cdegenerate oligonucleotide-primed PCRxe2x80x9d (DOP-PCR), with the additional advantage of species independence (Telenius (1992), Genomics 13, 718-725). DOP-PCR is based on the principle of priming from short sequences specified by the 3xe2x80x2-end of partially degenerate oligonucleotides used, during initial low annealing temperature cycles of the PCR protocol. Since these short sequences occur frequently, amplification of target DNA proceeds at multiple loci simultaneously.
DOP-PCR can be applied for generating libraries containing a high level of single-copy sequences, provided pure and a substantial amount of DNA of interest can be obtained, e.g. flow-sorted chromosomes, microdissected chromosome bands or isolated yeast artificial chromosomes (YACs). However, DOP-PCR seems to be not capable of providing a sufficient, uniform amplification of the DNA content of a single cell (Kuukasjxc3xa4rvi (1997), Genes, Chromosomes and Cancer 18, 94-101).
The sensitivity of PCR allows for the analysis of a specific target DNA in a single cell (Li (1988), Nature 335, 414-417). This led to the development of preimplantation genetic disease diagnosis using single cells from early embryos (Handyside (1989), Lancet 1, 347-349) and genetic recombination analysis using a single sperm (Cui (1989), Proc. Nati. Acad. Sci. USA 86, 9389-9393) or oocyte (Cui (1992), Genomics 13, 713-717). However, in all these cases the single cell can be analyzed only once for a given target sequence and independent confirmation of the genotype of any one cell is impossible.
A method called xe2x80x9cprimer-extension preamplificationxe2x80x9d (PEP) is directed to circumvent this problem by making multiple copies of the DNA sequences present in a single cell. PEP uses a random mixture of 15-base fully degenerated oligonucleotides as primers, thereby leading to amplification of DNA sequences from randomly distributed sites. It is estimated that about 78% of the genomic sequences in a single human haploid cell can be copied no less than 30 times (Zhang (1992), Proc. Natl. Acad. Sci. USA 89, 5847-5851). However, up to now, a complete and uniform amplification of a whole genome of a single cell has not been documented with methods such as PEP.
A method called representational difference analysis (RDA) is a subtractive DNA hybridization technique that discovers the differences between paired normal and tumor genomes (Lisitsyn (1993), Science 259, 946-951). The minimal amount of DNA needed for RDA shown is 3 ng, corresponding to ≈1xc3x97103 cells. However, only 70% of the genomic sequences can be reproducibly amplified by RDA (Lucito (1998), Proc. Natl. Acad. Sci. USA 95, 4487-4492). Therefore, a uniform and complete amplification of the entire genome of a single cell by representational difference analysis is not possible.
Therefore, considering the prior art described above, there is a demand for a method capable of substantially uniform and preferably complete amplification of genomic DNA, particularly from a single cell.
Thus, the technical problem consists in providing means and methods which comply with the needs as described above and which eliminate the above-mentioned disadvantages.
The solution to this technical problem is achieved by providing the embodiments characterized in the claims.
Accordingly, the present invention relates to a method for the amplification of DNA, comprising the steps of
(a) providing a sample comprising DNA;
(b) digesting the DNA to be amplified with a restriction endonuclease under conditions suitable to obtain DNA fragments of similar length, wherein said restriction endonuclease-is capable of providing 5xe2x80x2 overhangs wherein the terminal nucleotide of the overhang is phosphorylated or 3xe2x80x2 overhangs wherein the terminal nucleotide of the overhang is hydroxylated on said DNA fragments,
(c) annealing at least one primer to said DNA fragments wherein
(ca) (caa) simultaneously or subsequently, oligonucleotides representing a first primer are hybridized to said 5xe2x80x2 overhangs on said DNA fragments of step (b) and wherein oligonucleotides representing a second primer hybridize to 3xe2x80x2 overhangs generated by said first primer and wherein said first and second primer are of different length;
(cab) said second primer is ligated to said 5xe2x80x2 overhangs; and
(cac) said first primer is removed from said DNA fragments; or
(cb) (cba) simultaneously or subsequently, oligonucleotides representing a first primer wherein the nucleotide at the 5xe2x80x2 terminus is phosphorylated are hybridized to said 5xe2x80x2 overhangs on said DNA fragments of step (b) and wherein oligonucleotides representing a second primer hybridize with said first primer; and
(cbb) said first and second primer are ligated to said DNA fragments; or
(cc) (cca) oligonucleotides representing said primer are hybridized to said 3xe2x80x2 overhangs so that 5xe2x80x2 overhangs are generated; and
(ccb) said primer is ligated to recessed 5xe2x80x2 ends of said DNA fragments;
(d) filling in generated 5xe2x80x2 overhangs; and
(e) amplifying said DNA fragments with primers which are capable of hybridizing with the complementary strand of said primer(s) of step (c).
As used in accordance with the present invention, the term xe2x80x9cDNA fragments of similar lengthxe2x80x9d denotes fragments which, at a statistical level, have a size which is of comparable length. DNA fragments of comparable length are, for example, fragments of 50+/xe2x88x925 bp or of 4 kbp +/xe2x88x920.4 kbp. The length range of DNA fragments that is preferably generated is advantageously between about 50 bp and about 4 kbp. DNA fragments of greater or shorter length may be used as well, although they may be amplified or represented to a lesser extent than the above defined fragments. Preferably, the DNA fragments have a size of xe2x89xa63 kbp, more preferably said DNA fragments have an average length of about 1000 bp and particularly preferred are fragments of about 200-400 bp.
The term xe2x80x9c5xe2x80x2 overhangsxe2x80x9d as used herein means the 5xe2x80x2 phosphate group, provided e.g. by a staggered cleavage of DNA by restriction endonucleases, and denotes a single stranded overhanging 5xe2x80x2 end on DNA.
The term xe2x80x9cprimerxe2x80x9d as used herein refers to an oligonucleotide whether occurring naturally as in a purified restriction digest or produced synthetically. The primer is preferably single stranded for a maximum of efficiency in the method of the present invention, and is preferably an oligodeoxyribonucleotide. Purification of said primers is generally envisaged, prior to their use in the method of the present invention. Such purification steps can comprise HPLC (high performance liquid chromatography) or PAGE (polyacrylamide gel-electrophoresis), and are known to the person skilled in the art.
As used herein, the term xe2x80x9crestriction endonucleasexe2x80x9d refers to bacterial enzymes capable of cutting double stranded DNA at or near a specific nucleotide sequence. The term xe2x80x9cfilling inxe2x80x9d as used herein means a DNA synthesis reaction, initiated at 3xe2x80x2 hydroxyl ends, leading to a fill in of the complementary strand. This DNA synthesis reaction is Preferably carried out in presence of dNTPs (dATP, dGTP, dCTP and dTTP). Thermostable DNA polymerases such as Taq polymerases are generally used and are well known to the person skilled in the art.
The term xe2x80x9chybridized toxe2x80x9d in accordance with the present invention denotes the pairing of two polynucleotide strands by hydrogen bonding between complementary nucleotides. This hybridization includes hybridization wherein a primer is hybridized directly adjacent to said 5xe2x80x2 overhangs as well as hybridization wherein gaps between primers and protruding or receding ends of said DNA fragments are generated. For example, the method of the present invention can be conveniently carried out in the case that a gap is formed between said second primer and the 5xe2x80x2 end of the DNA fragment, to name an example, since this gap will be filled in by DNA polymerases, such as Taq polymerases, in embodiments where said Taq polymerase is added before or during annealing and hybridization steps.
Oligonucleotides, representing primers as used in the method of the present invention can be identified, obtained and tested according to the state of the art especially represented by computer based sequence analysis and laboratory manuals, e.g. Sambrook (Molecular Cloning; A Laboratory Manual, 2nd Edition, Cold Spring Harbour Laboratory Press, Cold Spring Harbour, N.Y. (1989)).
Furthermore, the setting of conditions for the above described steps of the method of the present invention is well within the skill of the artisan and to be determined according to protocols described, for example in Sambrook et al, loc. cit., or in the appended examples. Further examples of broader range hybridization conditions that can be employed in accordance with the invention are described, inter alia, in Ausubel, xe2x80x9cCurrent Protocols in Molecular Biologyxe2x80x9d, Green Publishing Associates and Wiley Interscience, N.Y. (1989), or Higgins and Hames (Eds) xe2x80x9cNucleic acid hybridization, a practical approachxe2x80x9d IRL Press Oxford, Washington DC, (1985).
From the above recited options of the following method for the amplification of DNA is preferred, said method comprising the steps of
(a) providing a sample comprising DNA;
(b) digesting the DNA to be amplified with a restriction endonuclease under conditions suitable to obtain DNA fragments of similar length, wherein said restriction endonuclease is capable of providing 5xe2x80x2 overhangs wherein the terminal nucleotide of the overhangs is phosphorylated or 3xe2x80x2 overhangs wherein the terminal nucleotide of the overhangs is hydroxylated on said DNA fragments;
(c) annealing at least one primer to said DNA fragments wherein
(ca) (caa) simultaneously or subsequently, oligonucleotides representing a first primer are hybridized to said 5xe2x80x2 overhangs on said DNA fragments of step (b) and wherein oligonucleotides representing a second primer hybridize to 3xe2x80x2 overhangs generated by said first primer and wherein said first and second primer are of different length;
(cab) said second primer is ligated to said 5xe2x80x2 overhangs; and
(cac) said first primer is removed from said DNA fragments;
(d) filling in generated 5xe2x80x2 overhangs; and
(e) amplifying said DNA fragments with primers which are capable of hybridizing with the complementary strand of said primer(s) of step (c).
In the case that the two primers are hybridized subsequently, the first primer after hybridization forms a 5xe2x80x2 overhang to which the second primer subsequently hybridizes.
The present invention is based on the surprising finding that the combination of the above mentioned steps leads to a substantially uniform and complete amplification of DNA, as demonstrated in Examples 2 and 3. In particular, the method of the invention has been exemplified as follows:
A DNA sample to be amplified can be obtained by isolating a single cell, i.e., for example, a bone marrow stroma cell, a single (tumorous) cell from peripheral blood, a single cell from umbilical vein blood or from a lymph node which is subjected to a digestion with a proteinase. After inactivation of the proteinase-activity, said sample DNA can be digested with a restriction endonuclease with four-nucleotide recognition site, like Msel, leading to DNA fragments of a similar length of about 200 to 400 bp.
The annealing and hybridization of a first and a second primer can be achieved by adding a primer comprising the nucleotide sequence as depicted in SEQ ID NO: 2, and a longer second primer comprising the nucleotide sequence as depicted in SEQ ID NO: 1. Said first primer can be additionally modified in that the last 3xe2x80x2 nucleotide of said primer is a dideoxy (dd)-nucleotide. The final concentration of primers in the following ligation reaction was 5 xcexcM. The ratio between primers used in the present invention to DNA to be amplified was in a range 3 Mio:1, more preferably said ratio was in the range of 300,000:1, most preferably said ration was in the range of 30,000:1. Furthermore, said primers can be pre-hybridized to each other before their addition to said DNA sample.
An annealing reaction was started at a temperature which served also to inactivate said restriction enzyme, i.e. 68xc2x0 C. Said second primer was ligated to said DNA fragments, in contrast to said first primer which is not ligated since no 5xe2x80x2 phosphate necessary for ligation was available. Therefore, the reaction temperature was gradually lowered to a temperature where such a ligation reaction can be carried out, i.e. 15xc2x0 C. Ligation was obtained by the addition of ATP and a T4-DNA-ligase, to said primers and DNA fragments. After ligation, said first primer was removed from said DNA fragments by a denaturation step, involving a change of temperature to a higher temperature (e.g. of about 68xc2x0 C.) wherein said primer dissociates from said DNA fragments. Said second primer remained ligated to said DNA fragments.
Resulting 5xe2x80x2 phosphate extensions on said DNA fragments were filled in by the addition of DNA polymerases, in the present case Taq and Pwo polymerase, in the presence of dNTPs (dATP, dCTP, dGTP and dTTP), under suitable conditions, as indicated in the examples.
The resulting mixture was then subjected to PCR amplification with said second primer, in a concentration of 1 xcexcM, as specified in examples 2 and 3.
The amplification product was then further analyzed as described in Example 3.
The method of the present invention is substantially independent from particular precautious measures that have to be observed in methods of the prior art. For example, with the method of the present invention, it is not necessary to extract or purify the DNA of interest (which could lead to losses of DNA) prior to amplification.
Thus, in contrast to the above-mentioned methods for DNA amplification, the method of the present invention can be carried out under amplification conditions which are convenient and optimal for the further use of different adaptor-ligated sequences of choice.
The method of the present invention for the first time allows the amplification of the entire genome of a single cell even from unextracted DNA samples. This enables e.g. the genomic analysis of individual isolated disseminated tumor cells, applying comparative genomic hybridization (CGH) to single cells. Therefore, this method provides, e.g., the means to identify the individual genetic changes in a single cell that might promote dissemination and ectopic growth of disseminated tumor cells with metastatic potential. The genomic profile of such single disseminated cells could provide useful information on whether certain clonal genotypes are associated with disseminative events.
Furthermore, the method of the present invention for the first time allows for the reproducible application of CGH to individual cells, whereas other protocols for xe2x80x9cwhole genome amplificationxe2x80x9d such as PEP, DOP-PCR and Alu-PCR do not provide homogenous staining patterns by CGH wherein the origin of test-DNA is a single cell, single chromosomes or parts thereof. This reproducible application of CGH on individual single cells can be explained, inter alia, by the fact that a non-degenerate primer is used that drastically reduces the complexity of primer binding sites of oligonucleotides hitherto used in the DOP and PEP techniques. These previously numerous different sites require equally numerous different specific PCR conditions impossible to achieve during the same reaction. Additionally, since the amplification method of the present invention does not depend on repetitive sequences within the genome (like IRS-PCR), it is possible to reliably amplify the genomic DNA of single cells from species where such repetitive sequences are less frequent or even non-existent.
In a preferred embodiment of the method of the present invention DNA which is amplified is the genome of a single cell or chromosomes or (a) fragment(s) thereof. It has surprisingly been found that the method of the present invention is particularly useful for the analysis of single cells such as disseminating tumor cells, cells obtained from a lymph node, peripheral blood cells, cells from bone marrow aspirates, cells from tumor biopsis, cells obtained from microdissected tissue, or the like. As shown in the appended examples, the method of the present invention is also useful for the analysis of the genome of any single cell (or chromosomes or fragments thereof) wherein said single cell is a rare event containing potentially interesting genetic information. Said single cell which is a rare event might be, inter alia, the cells described hereinabove or embryonic/fetal cells in the venous blood of the mother and the like. The inventive method is particularly useful for the assessment of clonal evolution events of genetic variants in complex (cell) populations, like, inter alia, the clonal evolution of single micro-metastatic cells isolated from peripheral blood, bone marrow, or the like.
The DNA content of a single diploid cell amounts only to 6-7 pg. In prior art DNA amplification methods, like DOP-PCR, at least 25 pg of DNA, corresponding to four diploid cells, are necessary for effective amplification of the entire DNA. However, as demonstrated in Examples 3, 4, 5 and 6 the method of the present invention provides the means to reliably amplify and analyze the entire genome of a single cell.
In another preferred embodiment of the method of the present invention said DNA is present in the form of one copy of a double stranded DNA sequence.
In a further preferred embodiment of the method of the present invention the numerical abundance of said DNA fragments is essentially maintained. As has been found in accordance with the present invention, the method of this invention is capable of reproducibly amplifying genomic sequences. Usually, 80%, preferably more than 90%, more preferably more than 95% and most preferably 99% or more of the genomic sequences can be amplified by the method of the present invention, and the amount of the amplification product for each of those genomic sequences substantially corresponds to their copy number in the genome, as demonstrated in example 4. Therefore, with the method of the present invention it is possible to amplify DNA of a given sample so that the ratio between genomic sequences remain the same before and after said DNA amplification. As discussed above, methods such as RDA (Lucito (1998), PNAS USA 95, 4487-4492) provide for amplifications wherein only 70% of genomic sequences are amplified. It is questionable whether the DNA fragments which are amplified by the prior art methods retain their relative numerical abundance.
In an additional preferred embodiment of the method of the present invention said method comprises, prior to step (a), the step (axe2x80x2) wherein said sample comprising DNA is digested with a proteinase and wherein, after the protein digest in step (bxe2x80x2), the proteinase is inactivated. Preferably, said proteinase is thermo-labil. Accordingly, said proteinase can be thermally inactivated in step (bxe2x80x2). In a preferred embodiment of the method of the present invention the said proteinase is Proteinase K.
In a further preferred embodiment of the method of the present invention said restriction endonuclease does not comprise any cytosine/guanine in its restriction site. It is well known in the art that genomic cytosine and guanine rests can be methylated, which might lead to a reduced enzymatic cut by restriction enzymes.
In a particularly preferred embodiment of the method of the present invention said restriction endonuclease recognizes a motif with four defined bases. Such endonucleases comprise enzymes which have four distinct nucleotides, e.g. Msel, in their recognition side as well as enzymes where an additional wobble base lies within the restriction side, like e.g. Apol. Preferably, said restriction endonuclease recognizes the consensus sequence TTAA.
In a most preferred embodiment of the method of the present invention said restriction endonuclease is Msel or an isoschizomer thereof. The convenience of using said restriction enzyme is demonstrated in example 2.
The first primer may be longer than the second primer, yet in another preferred embodiment, the present invention relates to the above described method wherein in step (caa) said second primer is longer than said first primer. As demonstrated in the examples, a convenient length difference is 8 to 12 bp.
In another preferred embodiment of the method of the invention the annealing temperature of said second primer in step (caa) is higher than the hybridizing temperature of said first primer to said second primer and said 5xe2x80x2 overhangs, as demonstrated in example 2.
In a particularly preferred embodiment of the method of the present invention in step (caa) said first primer comprises 11 or 12 nucleotides and said second primer comprises 21 nucleotides.
It is understood that in accordance with the method of the present invention said first primer in step (caa) or step (cba) is at least partially complementary to said second primer. In a particularly preferred embodiment said first and said second primer comprise a palindromic sequence.
In yet another preferred embodiment of the method of the present invention, the sequence of said first and said second primer is non-degenerate. As described above, other methods known to the person skilled in the art, such as DOP-PCR (Telenias (1992), Genomics 13, 718-725) are based on the use of degenerate oligonucleotides or partially degenerate primers. Such degenerate primers bear a high risk of self-annealing, thereby inhibiting themselves (each other) from binding to target sequences and resulting in the reduction of amplification efficiency.
In a yet more preferred embodiment, said first primer used in step (caa) has the sequence shown in SEQ ID NO: 2 and/or said second primer used in step (caa) has the sequence as shown in SEQ ID NO: 1.
In a further preferred embodiment of the method of the present invention, the last 3xe2x80x2 nucleotide of the first primer in step. (caa) of the above described method is modified, such that said primer cannot be elongated by polymerase activity (e.g. Taq polymerase activity). The person skilled in the art is well aware of such modifications and methods for producing such modified oligonucleotides. One of these modifications can be the addition of a dd-nucleotide at the 3xe2x80x2 end of the first primer in the above described step (caa).
In an additional preferred embodiment, said first and said second primer of the method of the present invention are hybridized to each other separately from said DNA fragments and are added to said DNA fragments after they hybridized to each other. The addition of the hybridized primers to said DNA fragments is effected prior to step (ca) or step (cb). Such a pre-hybridization of primers leads, inter alia, to a higher hybridization efficiency to said DNA fragments and interfering to chromosomal DNA can be avoided.
In another preferred embodiment of the method of the present invention essentially the whole nuclear genome of a single cell is amplified.
Usually, 80%, preferably more than 90%, more preferably more than 95% and most preferably 99% or more of the whole nuclear genome can be amplified by the method of the present invention.
In a particularly preferred embodiment of the method of the present invention said single cell is a chemically fixed cell. One option for chemically fixing a cell or tissue is formalin. Others are well known to the person skilled in the art.
The inventive method described herein can be applied to DNA of different sources, such as solid tumor DNA isolated from frozen sections and/or cryosections and/or paraffin embedded, formalin fixed specimens. For decades these tissue sections have been stored mainly for histopathological diagnosis. Single cells or small samples, comprising a limited amount of cells, from histopathological tissue can be screened for specific genetic changes and compared with other areas from the same tissue that may exhibit distinctly different histopathological features or, for control purposes, with areas of apparently normal tissue. Global screening of copy number sequence changes within a tumor genome from archival tissue material could increase the knowledge about cytogenetic alterations in solid tumors significantly. A direct comparison of these cytogenetic data with histological and histochemical results and clinical follow up data would become possible.
Furthermore, the method of the present invention may be used for the amplification of single-cell DNA which stems from microdissected and/or laser-microdissected (for example, laser microbeam microdissection preferably combined with laser pressure catapulting) material from, inter alia, cryosections, as shown in the appended examples.
In yet a more preferred embodiment of the method of the present invention steps (a) to (e) are carried out in one reaction vessel. This has the advantage that a potential template loss is avoided and, moreover, an additional opening and closing of the reaction vessel, which may involve contamination and is troublesome, is avoided.
As is evident to the person skilled in the art, the method of the present invention and/or the amplified DNA fragments obtained by the method of the present invention are particularly useful in diagnostic assays and as research tools. Said amplified DNA fragments are, inter alia, useful in areas and fields were only limited amounts of target-DNA is available, such as in forensic investigations (inter alia DNA-fingerprinting), in paleontology and/or in paleoarcheology. Furthermore, the method of the present invention and/or the amplified DNA fragments obtained therewith are particularly useful in preimplantation diagnosis on human and animal embryos. Said method and/or said amplified DNA fragments can furthermore be used, inter alia, in combination with chip-technologies, for identification assays for DNA of contaminating organisms in samples, such as food products or in blood- or liquor samples. Furthermore, the inventive method and/or the amplified DNA fragments obtained by said method may be useful in the detection of contaminating DNA in pharmaceutical compositions or diagnostic solutions.
The present invention therefore further relates to the use of the amplified DNA fragments obtained by the above described method in methods and techniques for DNA analysis. Such methods and techniques are routinely used in prenatal diagnosis, forensic medicine, pathogenic analysis or biological/biochemical research and are known to the person skilled in the art.
In a particularly preferred embodiment of the use of the present invention, the methods for DNA analysis are comparative genomic hybridization (CGH), representational difference analysis (RDA), analytical PCR, restriction enzyme length polymorphism analysis (RFLP), single strand conformation polymorphism analysis (SSCP), DNA sequence analysis, xe2x80x9closs of heterozygosityxe2x80x9d analysis (LOH), fingerprint analysis and/or fluorescence in situ hybridization (FISH).
A variety of techniques are now available for genome-wide screening of alterations in copy-number, structure and expression of genes and DNA sequences. These include molecular cytogenetic techniques (such as comparative genomic hybridization (CGH) and multicolor fluorescence in situ hybridization (M-FISH)), as well as molecular genetic techniques (such as representational difference analysis (RDA), differential display, serial analysis of gene expression (SAGE) and microarray techniques). CGH was the first molecular cytogenetic tool that allowed comprehensive analysis of the entire genome (Kallioniemi (1992), Science 258, 818-821). CGH allows for the screening for DNA sequence copy-number changes and provides a map of those chromosomal regions that are gained or lost in a DNA specimen. Because DNA copy-number alterations are of pathogenetic importance in cancer, most of the applications of CGH are in cancer research.
In CGH, which is based on a modified in situ hybridization, differentially labeled test (green) and reference (red) DNAs are co-hybridized to normal metaphase spreads. Copy-number differences between test and reference genomes are seen as green:red fluorescence intensity differences on the metaphase chromosomes. DNA gains and amplifications in the test DNA are seen as chromosomal regions with an increased fluorescence ratio, while losses and deletions result in a reduced ratio. An important contribution of CGH to cancer research has been in pinpointing putative locations of cancer genes, especially at chromosomal sites undergoing DNA amplification. A large number of subregional chromosomal gains and DNA amplifications have been discovered by CGH in some cancers. Because oncogenes and drug-resistance genes are known to be upregulated by DNA amplifications, it has been speculated that DNA amplification sites in cancer could pinpoint locations of novel genes with important roles in cancer progression. Tumor progression implies the gradual transition of a localized, slow growing tumor to an invasive, metastatic and treatment refractory cancer. This progression is thought to be caused by a stepwise accumulation of genetic changes affecting critical genes. By providing genome-scale information of clonal genetic alternations, CGH is extremely useful in the analysis of the biological basis of the tumor progression process. Two cancer specimen taken from the same patient at different stages of progression can be analyzed. For example, genetic changes that are not found in primary tumors, but do occur in their metastases could be informative in pinpointing genetic changes and genes with important roles in the metastatic progression. Metastatic relapse is caused by early dissemination of individual tumor cells, which leave their primary site and enter into the circulation prior to diagnosis and surgical removal of the primary tumor. The vast majority of these cells will be eliminated by the immune system or undergo apoptosis, while others will survive the perils of the circulation, invade tissues at a secondary site, and remain in a dormant stage for years before they finally grow to metastases. This early stage of metastasis formation (minimal residual disease), when tumor cells are few and dispersed, represents the xe2x80x9cAchilles"" Heelxe2x80x9d of cancer, being a promising target for the development of new therapeutic approaches to prevent clinical metastasis. Therefore, in order to screen individual tumor cells by methods like CGH, it is desirable to uniformly and accurately amplify the whole genome of such a cell. Accordingly, the above described method is particularly useful for screening individual tumor cells by CGH and therefore allowing early diagnosis of e.g. neoplastic disorders or patients susceptible to such disorders. The term xe2x80x9cneoplastic disordersxe2x80x9d is intended to mean the whole spectrum from initiation of malignant transformation in a single cell to advanced cancer disease, including distant solid metastasis.
Based on CGH and DOP-PCR the minimal amount of target DNA so far needed for a reproducible amplification is 50 pg, corresponding to 8 diploid cells (Speicher (1993), Hum. Mol. Genet. 11, 1907-1914). Smaller amounts of genomic DNA could not be reproducibly amplified. Using the method of the present invention a further reduction of the amount of DNA necessary for a single test is possible.
The method of the present invention not only provides for example uniformly amplified DNA for the subsequent use in CGH, but also allows the reliable uniform amplification of even smallest quantities of DNA for further techniques and methods, wherein samples contain only small amounts of DNA. For example, in forensic science, DNA typing procedures have become increasingly important in the last few years (Lee (1994), Am. J. Forensic Med. Pathol. 15, 269-282): PCR and RFLP analysis, also called fingerprint analysis, are carried out with only minute available quantities of DNA found in sperm, blood traces or individual cells and the like.
Another important application of the presented method is prenatal diagnosis using embryonic or fetal cells in maternal blood. The ability to use embryonic or fetal cells enriched from maternal blood of pregnant women for prenatal diagnosis of chromosomal abnormalities has been a long-sought goal for those pursuing a non-invasive alternative to current methods, such as amniocentesis or chorionic villus sampling. The localization and identification of novel disease genes allows for mutation analysis or linkage studies on fetuses at risk for single gene disorders or chromosomal abnormality etc. The method of the present invention improves the accuracy as well as applicability of methods for the diagnosis of preimplantation genetic disorders or for the diagnosis on fetal cells isolated from maternal blood, whereby analyses can be performed on a single cell level, thus abolishing the need for preceding enrichment of cells. As demonstrated in the appended examples, the method of the present invention provides for the reliable amplification of DNA from a single fetal or embryonic cell isolated from maternal blood, inter alia, from the umbilical vein blood.
The present invention further relates to a kit comprising at least one primer and/or a first and/or a second primer as defined above. Advantageously, the kit of the present invention further comprises, besides said primer and/or the primers, optionally, proteinases, restriction enzymes, DNA-ligases (such as T4-DNA-Ligase), DNA-polymerases (such as Taq polymerase), Pwo polymerase, and/or ThermoSequenase, as well as (a) reaction buffer(s) and/or storage solution(s). Furthermore, parts of the kit of the invention can be packaged individually in vials or in combination in containers or multicontainer units. As it has been usefully demonstrated in the examples, a Proteinase K-digestion, a four-cutting restriction-endonuclease and primer(s) as defined above are suitable for the method of the invention. Thus, the kit of the invention preferably comprises Proteinase K, a four-cutting restriction endonuclease (such as Msel), Taq and/or Pwo polymerase, primer(s) as defined above, and/or T4-Ligase. The kit of the present invention may be advantageously used for carrying out the method of the invention and could be, inter alia, employed in a variety of applications referred to above, e.g. in diagnostic kits or as research tools. Additionally, the kit of the invention may contain means for detection suitable for scientific and/or diagnostic purposes. The manufacture of the kits follows preferably standard procedures which are known to the person skilled in the art.
Furthermore, the present invention relates to the use of a first and/or second primer as defined above for the preparation of a kit for carrying out the method of the present invention.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word xe2x80x9ccomprisexe2x80x9d, and variations such as xe2x80x9ccomprisingxe2x80x9d and xe2x80x9ccomprisingxe2x80x9d, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step.